Angiogenesis promotes tumor progression and enables metastatsis. Previous studies focused on the function of tumor signals that activate endothelial cell responses. However, the function of endothelial cell signals that directly elicit tumor cell responses remain uncharacterized. The goal of this proposal is to identify and characterize novel, endothelial-derived molecular regulators of tumor cell growth and invasion. Our preliminary studies showed that conditioned media from wild-type, but not EphA2-deficient, endothelial cells promotes tumor cell growth and motility in culture. Co-transplanted wild-type, but not EphA2-deficient, endothelial cells enhanced tumor cell proliferation in the absence of blood flow in vivo. Microarray analysis of wild-type and EphA2-deficient endothelial cells revealed that mRNA encoding slit2 is elevated in EphA2-deficient endothelial cells. Slit2 is a secreted ligand for the roundabout (Robo) receptor family that functions in neural development, and was more recently shown to negatively regulate angiogenesis and tumor progression. In addition we observed the absence of slit2 protein in EphA2-positive blood tumor vessels within a significant number of human breast cancer samples, and also found that ephA2 correlates negatively, while slit2 correlates positively, with overall survival in human breast cancer patients. This led us to hypothesize that EphA2 downregulates slit2 ligand to facilitate endothelial cell-induced tumor cell proliferation, migration, and progression, as well as angiogenesis. This research will enhance our understanding of how seemingly normal tissue in the microenvironment, such as endothelium, that surrounds the tumor facilitates growth, invasion, and metastasis, an NCI priority for FY2010 and beyond. Investigation will be completed at Vanderbilt University School of Medicine in the context of the Vanderbilt-Ingram Comprehensive Cancer Center facilities, which supports a host-tumor interaction focused research environment ideally suited for the molecular, biochemical, genetic, and imaging analyses involved in this research.